Genetic instability, a hallmark feature of cancer, often arises from chromosome segregation errors during mitosis in part due to deregulation of certain cancer-associated genes. Over the past decade, Hec1 (Highly Expressed in Cancer 1), originally discovered by us in 1997, has been increasingly recognized as a key regulator in mitotic processes including kinetochore-microtubule attachment, Mad2-dependent spindle assembly checkpoint activation and spindle assembly. Importantly, overexpression of Hec1 associates with poor prognosis of primary breast cancers in human and is also found to initiate tumor formation in a mouse model, thus stipulating Hec1 as an oncogene. Recent work from our lab has shown that targeting the Hec1/Nek2 mitotic pathway with a small molecule INH1 efficiently retards the breast cancer cell growth in culture and in animals, demonstrating that Hec1 may serve as a novel molecular target for cancer intervention. In this application, we propose to refine and optimize the lead compound INH1 to generate analogues feasible for potential clinical application as well as to elucidate the detailed molecular and cellular mechanism of how the INH1 inhibitor works. Two major specific aims are proposed in this application: first is to design, synthesize, and biologically evaluate the second and third generations of INH1 analogues; second is to elucidate molecular and cellular mechanisms of the most active INH analogues. We are in an excellent position to further generate derivatives with nanomolar potency and to dissect the detailed mode-of-action of INH compounds. Accomplishing the objectives outlined in this study will represent an important milestone toward the translation of this potential drug into clinical application.